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Genome Biology 2006, 7:R51 comment reviews reports deposited research refereed research interactions information Open Access 2006Polavarapuet al.Volume 7, Issue 6, Article R51 Research Identification, characterization and comparative genomics of chimpanzee endogenous retroviruses Nalini Polavarapu, Nathan J Bowen and John F McDonald Address: School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230, USA. Correspondence: John F McDonald. Email: john.mcdonald@biology.gatech.edu © 2006 Polavarapu et al.; BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chimpanzee endogenous retroviruses<p>The identification and characterization of 42 families of chimpanzee endogenous retroviruses and a comparison to their human orthologs is described.</p> Abstract Background: Retrotransposons, the most abundant and widespread class of eukaryotic transposable elements, are believed to play a significant role in mutation and disease and to have contributed significantly to the evolution of genome structure and function. The recent sequencing of the chimpanzee genome is providing an unprecedented opportunity to study the functional significance of these elements in two closely related primate species and to better evaluate their role in primate evolution. Results: We report here that the chimpanzee genome contains at least 42 separate families of endogenous retroviruses, nine of which were not previously identified. All but two (CERV 1/ PTERV1 and CERV 2) of the 42 families of chimpanzee endogenous retroviruses were found to have orthologs in humans. Molecular analysis (PCR and Southern hybridization) of CERV 2 elements demonstrates that this family is present in chimpanzee, bonobo, gorilla and old-world monkeys but absent in human, orangutan and new-world monkeys. A survey of endogenous retroviral positional variation between chimpanzees and humans determined that approximately 7% of all chimpanzee-human INDEL variation is associated with endogenous retroviral sequences. Conclusion: Nine families of chimpanzee endogenous retroviruses have been transpositionally active since chimpanzees and humans diverged from a common ancestor. Seven of these transpositionally active families have orthologs in humans, one of which has also been transpositionally active in humans since the human-chimpanzee divergence about six million years ago. Comparative analyses of orthologous regions of the human and chimpanzee genomes have revealed that a significant portion of INDEL variation between chimpanzees and humans is attributable to endogenous retroviruses and may be of evolutionary significance. Background Retrotransposons are the most abundant and widespread class of eukaryotic transposable elements. For example, >30% of the mouse genome [1], >50% of the maize genome [2] and >60% of the human genome [3] are composed of ret- rotransposon sequences. This group of transposable elements is made up of short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs) and long termi- nal repeat (LTR) retrotransposons/endogenous retroviruses, all of which replicate via reverse transcription of an RNA Published: 28 June 2006 Genome Biology 2006, 7:R51 (doi:10.1186/gb-2006-7-6-r51) Received: 29 March 2006 Revised: 23 May 2006 Accepted: 25 May 2006 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/6/R51 R51.2 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, 7:R51 intermediate [4]. The biological significance of retrotrans- posons ranges from their contribution to mutation (for exam- ple, [5]) and disease (for example, [6,7]) to their role in gene and genome evolution (for example, [8-10]). The recent sequencing of the chimpanzee genome has pro- vided an unprecedented opportunity to not only compare the full complement of retrotransposons in two closely related primate species but to gain insight into the role these ele- ments may have played in human evolution. We have com- bined the use of an LTR retrotransposon search algorithm, LTR_STRUC [11], with a systematic series of iterative TBLASTN searches to identify the endogenous retroviruses present in the Ensembl chimpanzee database [12]. Since LTR_STRUC searches for LTR retrotransposons/endog- enous retroviruses based on structure rather than homology, elements are often identified that go undetected in traditional BLAST searches (for example, [11]). LTR_STRUC is designed specifically to find full-length LTR retrotransposons/endogenous retroviruses, that is, ones hav- ing two LTRs and a pair of target site duplications (TSDs) [11]. Thus, we complemented our search by using reverse tran- scriptase (RT) sequences from LTR_STRUC-identified ele- ments as query sequences in an iterative series of TBLASTN searches. This allowed us to identify structurally aberrant ele- ments not directly detected by LTR_STRUC. Finally, a series TBLASTN searches were carried out using, as query sequences, previously reported human RT sequences for which orthologues were not identified by our previous two searches. Results and discussion The chimpanzee genome contains at least 42 families of endogenous retroviruses Using the procedure described above, we identified a total of 425 full-length chimpanzee endogenous retroviruses. This is certainly an underestimate of the number of endogenous ret- roviruses in the chimpanzee genome because we consciously excluded any sequences that could not be unambiguously identified as an endogenous retrovirus. The majority of these endogenous retroviruses (395/425 or 93%) were identified directly by LTR_STRUC or by homology to LTR_STRUC- identified elements. ClustalX [13] was used to build a multiple alignment of the RT domain of these 425 elements together with the RT domains of 16 previously described LTR retrotransposons/retrovi- ruses representative of the three major classes of retroviral elements (Table 1). Phylogenetic analysis of the RT regions of the 425 full-length elements revealed the presence of at least 42 independent lineages of endogenous retroviruses in the chimpanzee genome that we here define as families (Figure 1). Non-autonomous endogenous retroviruses are elements that lack an RT open reading frame (ORF) and are required to utilize RT activity from autonomous, full-length endogenous retrovirus in order to replicate. Many of the chimpanzee endogenous retrovirus families contain truncated, non- autonomous as well as full-length elements. Of the 42 families of chimpanzee endogenous retroviruses identified in this study, 40 were found to have orthologues in the human genome, including 9 that were identified in this study for the first time [14] (see Additional data file 1). Two previously identified chimpanzee endogenous retrovirus families do not have human orthologues (Table 2). Consistent with the consensus nomenclature used for human endogenous retroviruses (HERV) [4], we here refer to the chimpanzee endogenous retroviral families by the acronym CERV (for chimp endogenous retrovirus). Distinct families are indicated by number (for example, CERV 1 to CERV 42). In the single instance where the CERV acronym refers to a previously named element/family, we include the pre-exist- ing nomenclature as well (CERV 1/PTERV1). In those cases where a CERV family has an orthologue in humans, the name of the orthologous HERV family is given in parentheses (for example, CERV 3(HERVS71)). Endogenous retroviral families of the chimpanzee genome LTR retrotransposons and retroviruses are grouped into three major classes [15]. Class I contains elements related to the gammaretroviruses (for example, Moloney murine leuke- mia virus (MuLV; accession no. AF033811), gibbon ape leukemia virus (GALV; accession no. M26927) and feline leukemia virus (FeLV; accession no. M18247)). Class II ele- ments are related to betaretroviruses (for example, mouse mammary tumor virus (MMTV; accession no. NC_001503), rabbit endogenous retrovirus (RERV; accession no. AF480925)). Class III elements are distantly related to spu- maviruses (for example, human foamy virus (HFV; accession no. Y07725), feline foamy virus (FeFV; accession no. AJ223851)). Of the 42 chimpanzee families identified in our study, 29 belong to class I, 10 to class II and 3 to class III (Fig- ure 1). While there is a precedence for classifying human endog- enous retroviruses into families based on their tRNA primer- binding sites (for example, HERV K (lysine tRNA binding site)) [4], we find that such groupings do not accurately reflect the phylogenetic groupings of CERVs. For example, some members of the CERV 21 family have a proline tRNA binding site whereas other members of this same family uti- lize threonine tRNA as a primer. Conversely, phylogenetically divergent CERV families may share the same tRNA binding site (for example, members of the CERV 27 (HERV I) and CERV 30 (HERVK10) have lysine tRNA binding sites) (Table 2). Thus, primer binding sites appear to be an evolutionarily labile feature and thus not a reliable indicator of phylogenetic relationships among chimpanzee endogenous retroviruses. A http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. R51.3 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2006, 7:R51 similar conclusion has been drawn for LTR retrotransposons in Caenorhabditis elegans [16]. Full-length CERVs are typically between 7,000 and 10,000 base-pairs (bp) in length. Consistent with what has been reported for LTR retrotransposons/endogenous retroviruses in other species [17-19], CERV target site duplications (TSDs) range in size from 4 to 6 bp in length. With the exception of a few mutated copies, CERVs have the same canonical dinucle- otides terminating the LTRs as have been reported for LTR retrotransposons/endogenous retroviruses in other species (TG/CA) [17-19]. CERV LTRs are typically 400 to 600 bp in length, although some LTRs are variant in size due to INDELs. For example, the LTRs of a member of the CERV 4 Unrooted RT based neighbor joining tree of three classes of chimpanzee endogenous retroviruses: class I, CERV1 to CERV29; class II, CERV30 to CERV 39; class III, CERV 40 to CERV 42Figure 1 Unrooted RT based neighbor joining tree of three classes of chimpanzee endogenous retroviruses: class I, CERV1 to CERV29; class II, CERV30 to CERV 39; class III, CERV 40 to CERV 42. Bootstrap values are shown for each of the families. RT sequences from species other than chimpanzee, listed in Table 1, are included for comparison. 100 100 100 100 100 100 100 100 100 RERV GH G18 SRV -1 MMTV RSV 100 HFV FeFV 100 100 100 HIV BLV 100 78 100 100 91 100 100 80 86 100 100 100 100 BaEV FEL V MuLV PERV MDEV GALV KoR V 100 100 84 100 99 56 97 100 100 100 100 100 100 100 100 100 CERV 1 CERV 2 CERV 3 CERV 4 CERV 5 CERV 6 CERV 7 CERV 8 CERV 9 CER V 10 CER V 11 CERV 12 CERV 13 CERV 14 CER V 15 CERV 16 CER V 17 CER V 18 CERV 19 CERV 20 CERV 21 CERV 22 CERV 23 CERV 24 CERV 25 CERV 26 CERV 27 CERV 28 CERV 29 CERV 30 CERV 31 CERV 32 CERV 33 CERV 34 CERV 35 CERV 36 CERV 37 CERV 3 8 CERV 39 CERV 40 CERV 41 CERV 42 Class II Class III Class I 0.1 R51.4 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, 7:R51 (HERV 3) family are 1,591 bp in length due to the insertion of an Alu element at some point in the evolutionary history of this lineage. The following is a more detailed characterization of the three classes of CERVs. Class I: families 1 to 29 The CERV families 1 through 29 group with the class I retro- viruses (Figure 1; Additional data file 2). The average size of full-length class I CERVs is 8,443 bp. These elements range in size from 2,268 to 13,135 bp in length. Much of this variation is due to INDELs associated with non-functional elements. The average size of LTRs associated with full-length class I CERV elements is 544 bp (range 195 to 1,591 bp). Class I CERV elements display considerable variation in their tRNA binding sites, even within families (Table 2). The most fre- quently used tRNA primer for class I CERV families (28%) is proline tRNA. Because the LTRs of endogenous retroviruses are synthesized from a single template during reverse transcription, they are identical at the DNA sequence level upon integration [4]. Using the primate pseudogene nucleotide substitution rate of 0.16% divergence per million years [20,21], the relative inte- gration time or age of CERV elements can be estimated from the level of sequence divergence existing between the element's 5' and 3' LTRs. The Jukes-Cantor model was used to correct for the presence of multiple mutations at the same site, back mutations and convergent substitutions [22]. Although caution must be taken when using LTR divergence to estimate the age of individual elements because of con- founding processes such as recombination and conversion, (for example, [23,24]), the method is able to provide useful age estimates, at least to a first approximation (for example, [25]). Using this method, we estimate that the age of full- length class I CERV elements ranges from 0.8 to 82.9 million years (MY). Full length elements representing at least three class I CERV families, CERV 1/PTERV1, CERV 2 and CERV 3 (HERVS71) have been recently transpositionally active as indicated by the presence of an unoccupied pre-integration site at the corre- sponding locus in humans. Inconsistent with this view is the fact that one of the chimpanzee-specific CERV 3 (HERVS71) insertions located on the Y chromosome displays an atypi- cally high level of LTR-LTR sequence divergence (9%), indic- ative of it having inserted about 28 million years ago (MYA). However, the clear absence of this insert, both in the sequenced human genome (pre-integration site in tact) and in the genomes of several randomly sampled ethnically and geographically diverse humans (data not shown), indicates that this element most likely inserted after the chimpanzee- human divergence (about 6 MYA) and that the exceptionally high level of LTR-LTR sequence divergence is due to an inter- element recombination or conversion event [23,24]. All other class I CERV elements are much older and have not been reproductively active since well before chimpanzees and humans diverged from a common ancestor. Class II: families 30 to 39 The CERV families 30 through 39 group with class II retrovi- ruses (Figure 1; Additional data file 3). All Class II CERV fam- ilies have orthologues in humans. The average size of full- length class II CERVs is 7,670 bp. This class of CERV ele- ments range in size from 2,564 to 12,803 bp in length. As with Table 1 Previously characterized RT sequences from a variety of species used for comparison in phylogenies Name Name of retrovirus Accession number RERV Rabbit endogenous retrovirus AF480925 GH-G18 Golden hamster intracisternal A-particle H18 GNHYIH SRV-1 Simian SRV-1 type D retrovirus M11841 MMTV Mouse mammary tumor virus NC_001503 RSV Rous sarcoma virus AF052428 HFV Human foamy virus Y07725 FeFV Feline foamy virus AJ223851 HIV-1 Human immunodeficiency virus 1 K03454 BLV Bovine leukemia virus K02120 BaEV Baboon endogenous virus X05470 FELV Feline leukemia virus M18247 MuLV Moloney murine leukemia virus AF033811 PERV Porcine endogenous retrovirus AF038601 MDEV Mus dunni endogenous virus AF053745 GALV Gibbon ape leukemia virus M26927 KoRV Koala type C endogenous virus AF151794 Also see Figure 1 and Additional data files 2-4 http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. R51.5 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2006, 7:R51 class I elements, much of the size variation among class II ele- ments is due to INDELs associated with non-functional elements. The average size of LTRs associated with full-length class II CERV elements is 544 bp (range 243 to 1,139 bp). Consistent with the fact that class II CERVs are orthologous to human HERV K elements, all but one family of class II CERV elements have lysine tRNA binding sites. The sole exception, CERV 39 (HERV K22), has a methionine tRNA binding site (Table 2). It has recently been proposed that HERV K22 be renamed HERV M to reflect its distinct primer binding site [26]. Unlike the other class II CERV elements, the CERV 39 (HERV K22) family clusters closely with the betaretrovirus (MMTV, SRV-1) (Figure 1; Additional data file 3). Table 2 Representative sequences from each family of chimpanzee endogenous retroviruses Family name: chimp family (orthologous human family) tRNA primer Location on chromosome (chromosome no: position) 5' and 3' LTR % identity Length of 5'/3' LTRs (bp) Target site repeats Dinucleotides Element length (bp) CERV 1/PTERV1 Pro 8:62466629 62474817 99.7 409/409 GTAT/GTAT TG/CA 8,189 CERV 2 Pro 1:53871490 53880190 98.8 497/486 GTGA/GTGA TG/CA 8,338 CERV 3 (HERVS71) Thr 7:45002408 45013133 90 528/524 AGGC/AGGC TG/CA 10,726 CERV 4 (HERV3) Arg 6:65506183 65515842 91.7 643/592 TATA/TATA TG/CA 9,660 CERV 5 (HERV15) Thr 20:22151622 22161290 90 495/500 TTTT/TTTT TG/CC 9,669 CERV 6 (HERV 1*) Thr 8:43017710 43027603 92 511/512 CCAC/CCAC TG/CA 9,697 CERV 7 (Harlequin) Glu 14:49265903 49274634 90 477/470 ATAAAT/ATAAAT TG/CA 8,745 CERV 8 (HERVE) Glu 4:29336385 29342031 92.3 354/355 AACA/AACA TG/CA 5,647 CERV 9 (HERV 2*) His 1:74420643 74424449 74.5 318/315 CTTTT/CTTTT TG/CA 3,807 CERV 10 (HERVH48) Phe 1:162967211 16293841 86 404/401 ATTCT/ATTCT TG/CA 6,640 CERV 11 (HERV-H) His 13:88231927 88241255 91 367/367 TGTTA/TGTTA TG/CA 9,329 CERV 12 (HERVFH19) ND 1:9084130 9093376 88 412/421 ND ND 9,236 CERV 13 (PRIMA4) Arg X:81351532 81361722 86 627/617 CCTC/CCTC TG/CA 10,191 CERV 14 (HERV 5*) Leu, Arg 3:58020412 58027601 83 420/430 CACT/CACT TG/CA 7,198 CERV 15 (HERV-P) Pro, Val 6:89330483 89339138 87 634/625 ATACC/ATACT TG/CA 8,500 CERV 16 (HERV-17) Gln, Arg 4:55955342 55963662 89 775/760 CCTT/CCTT TG/CA 8,329 CERV 17 (HERV30) Leu, Arg 5:121436599 121446300 89 698/700 AAAG/AAAG TG/CA 9,702 CERV 18 (HERV 9) Arg, Lys, Pro 5:39234784 39242752 87 423/449 GGAG/GGAG TG/CA 7,966 CERV 19 (PABL_B) Arg, Leu 7:75174020 75182429 83 671/667 AGAG/AGAG TG/CA 8,410 CERV 20 (HERVP71A) Pro X:121795847 121803454 85 464/458 TTTTC/TTTTC TG/CA 7,608 CERV 21 (HERV 4*) Thr, Pro 2:14653540 14661848 91 361/363 ATGA/ATGA TG/CA 8,321 CERV 22 (HERV 6*) Thr 16:84359558 84368114 86 434/435 ND AG/CT 8,557 CERV 23 (HERV 7*) Pro 17:39042670 39052505 88 582/575 AGAC/AGAC TG/CA 9,836 CERV 24 (HERV 10*) Pro 17:27621756 27630879 87 429/431 TAAT/TAAT TG/CA 9,132 CERV 25 (HERV 11*) Pro 1:32316520 32325874 84 613/621 GCAAA/GCAAA TG/CA 9,480 CERV 26 (HERV 12*) ND 2:171938873 171948150 93 509/506 AATT/ACTT TG/CA 9,279 CERV 27 (HERVI) Lys 5:122623439 122630725 89.9 497/506 CAGT/CAGT CG/CA 7,287 CERV 28 (HERVIP10F) Ala 23:41095392 41106316 95.6 494/496 TACT/TACT TG/CA 10,925 CERV 29 (HERVG25) ND 6:151527383 151531731 84 220/226 ND ND 4,349 CERV 30 (HERVK10) Lys 10:4757815 4766975 99.4 961/958 ATTAT/ATTAT TG/CA 9,161 CERV 31 (HERVK14) Lys 9:74609341 74617943 92 623/618 CAATG/CAATG TG/CA 8,603 CERV 32 (HERVK14C) Lys 12:84993042 85001650 92 583/583 ND TG/CA 8,609 CERV 33 (HERVK(C4)) Lys 1:134530572 134538281 94 541/547 ATTAAG/ATTAAG TG/CA 7,710 CERV 34 (HERVK9) Lys X:58520547 58526579 93.4 510/508 GCCTAG/GCCTAG TG/CA 6,033 CERV 35 (HERVK13) ND 19:41852728 41865530 83 812/818 ND ND 12,803 CERV 36 (HERVK11D) Lys 5:123901088 123908580 91 865/874 ATAAAT/ATAAAT TG/TA 7,493 CERV 37 (HERVK11) Lys 2:81402412 81411338 95.7 1,079/1,079 ATAAAA/ATAAAA TG/CA 8,927 CERV 38 (HERVK3) Lys 5:26871940 26880204 95.2 428/431 GGTAAA/GGTGAA TG/CA 8,265 CERV 39 (HERVK22) Met 5:103484321 103492150 85 477/497 GTTCTT/GTTCTT TG/CA 7,830 CERV 40 (HERV S) Ser 1:147219818 147226527 85 325/329 CCATC/CCATC TG/CA 6,710 CERV 41 (HERV16) ND X:103812023 103815002 ND ND ND ND 2,980 CERV 42 (HERVL) Leu 3:83740925 83746481 82 445/458 ATAAT/ATAAT TG/CA 5,547 *Families submitted to Repbase. ND, not determined. R51.6 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, 7:R51 The estimated age of full-length class II CERV elements ranges from 2 to 97 MY. A member of only one class II family, CERV 30 (HERV K10), has been transpositionally active since the divergence of chimps and humans from a common ances- tor. The LTR sequence identity of one of the identified CERV 30 (HERVK10) elements is 99.4%, indicating that this ele- ment inserted into the chimpanzee genome about 2 MYA. We have verified that this CERV 30 (HERV K10) insertion is absent in humans (Figure 2). It has been previously reported [27,28] and we found in our INDEL analysis (see below) that at least 8 full-length copies of CERV 30 orthologue HERV K10, inserted into the human genome after the divergence of chimpanzees and humans from a common ancestor. In addi- tion, two CERV 30 (HERV K10) insertion polymorphisms have been identified in human populations [29]. Thus, CERV 30 (HERV K10) family members and their human ortho- logues have been transpositionally active in both human and chimpanzee lineages since these species diverged from a com- mon ancestor about 6 MYA. CERV 36 (HERV K11D) is the second oldest family of class II CERV elements. We estimate that CERV 36 (HERV K11D) elements have not been transpositionally active for about 25 MY. We found that several members of the CERV 36 (HERV K11D) display the same deletion within the gag-pol regions of their genomes, suggesting that this deletion occurred prior to their transposition. Thus, this subfamily of CERV 36 (HERV K11D) elements comprised, at one time, non-autonomous elements and acquired essential replicative functions in trans. Class III: families 40 to 42 The CERV families 40 (HERV S), 41 (HERV 16) and 42 (HERV L) group with class III retroviruses and are related to spumaviruses [4] (Figure 1; Additional data file 4). All class III CERV families have orthologues in humans. The average size of full-length class III CERVs is 6,758 bp. This class of CERV elements range in size from 2,980 to 13,271 bp in length. Again, much of this size variation is due to INDELs in this uniformly non-functional class of CERV elements. The average size of LTRs associated with full-length class III CERV elements is 446 bp (range 254 to 831 bp). CERV 40 ele- ments have a serine tRNA binding site while CERV 42 ele- ments have a leucine tRNA binding site (Table 2). Due to sequence ambiguities, we were unable to determine the tRNA binding site for CERV 41 elements (Table 2). Class III CERV elements are the oldest group of endogenous retroviruses in the chimpanzee genome. The estimated age of these elements ranges from 30 to 145 MY. Two CERV families have no human orthologues CERV 1/PTERV1 With more than 100 members, CERV 1/PTERV1 is one of the most abundant families of endogenous retroviruses in the chimpanzee genome. CERV 1/PTERV1 elements range in size from 5 to 8.8 kb in length, are bordered by inverted terminal repeats (TG and CA) and are characterized by 4 bp TSDs (Table 2). The LTRs of the CERV 1/PTERV1 family of ele- ments range from 379 to 414 bp in length. CERV 1/PTERV1 elements have a proline tRNA primer binding site (Table 2). LTR sequence identity among CERV 1/PTERV1 elements ranges from 97.1% to 99.7%. Phylogenetic analysis of the LTRs from full-length elements of CERV 1/PTERV1 members indicated that this family of LTRs can be grouped into at least two subfamilies (bootstrap value of 99; Figure 3). The age of each subfamily was estimated by calculating the average of the pairwise distances Insertion of a member of the CERV 30 (HERVK10) family in chimpsFigure 2 Insertion of a member of the CERV 30 (HERVK10) family in chimps. The insertion occurred in the LINE element present in chromosome 10 of the chimpanzee genome. The orthologous LINE element is present in chromosome 12 in humans. In chimpanzees target site duplications (ATTAT) are identified. A single copy of TSD (ATTAT, the pre-integration site) is found inside the LINE element in humans. The LTRs of the element are 99.4% identical. LTR ATTAT LTR gag pol env A TTA T Preintegration site (ATTAT) Human Chr 12 Chimp Chr 10 LINE LINE LINE CERV 30 (HERVK10) http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. R51.7 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2006, 7:R51 between all sequences in a given subfamily. The estimated ages of the two subfamilies are 5 MY and 7.8 MY, respectively, suggesting that at least one subfamily was present in the line- age prior to the time chimpanzees and humans diverged from a common ancestor (about 6 MYA). This conclusion, however, is inconsistent with the fact that no CERV 1/ PTERV1 orthologues were detected in the sequenced human genome. Moreover, we were able to detect pre-integration sites at those regions in the human genome orthologous to the CERV 1/PTERV1 insertion sites in chimpanzees, effectively eliminating the possibility that the elements were once present in humans but subsequently excised. Consistent with our findings, the results of a previously published Southern hybridization survey indicated that sequences orthologous to CERV 1/PTERV1 elements are present in the African great apes and old world monkeys but not in Asian apes or humans [30]. These results suggest that some members of the CERV 1/PTERV1 subfamily entered the chimpanzee genome after the split from humans through exogenous infections from closely related species and subsequently increased in copy number by retrotransposition. The unexpectedly high level of LTR-LTR divergence could be due to variation accumulated during the viral transfer [31] or possibly due to an inter-ele- ment recombination or conversion event subsequent to inte- gration. Similar results were obtained when only the solo LTRs or both solo LTRs and LTRs from full-length elements were used in constructing the phylogenetic trees (Additional data files 5 and 6). We found that a number of CERV 1/PTERV1 elements with high (>99%) LTR-LTR sequence identity have large (1 to 2 kb) deletions within the RT encoding region of their genomes. It is likely that these are non-autonomous elements that have inserted relatively recently by acquiring RT functions in trans, presumably from autonomous CERV 1/PTERV1 ele- ments. Instances of recently inserted LTR retrotransposons/ endogenous retroviruses lacking RT-encoding functions have previously been detected in the genomes of humans [32] and other species of both plants [18,33] and animals (for example, [16]). CERV 2 This is the second family of chimpanzee endogenous retrovi- ruses with no orthologue in the human genome. We identified ten solo LTRs and eight full-length copies of CERV 2 elements in the chimpanzee genome although, because of incomplete sequencing, we could identify the LTRs for only four of the eight full-length elements. CERV 2 elements are typically larger than CERV 1/PTERV1 elements, ranging in size from 8 to 10 kb in length. CERV 2 elements are bordered by inverted terminal repeats (TG and CA), have 4 bp TSDs (Table 2) and a proline tRNA primer binding site (Table 2). The LTRs of the CERV 2 family of elements range from 486 to 497 bp in length. Based on their LTR sequence identity (98.07% to 99.6%), we estimate that full-length CERV 2 elements were transpositionally active in the chimpanzee genome between 1.3 and 6.0 MYA. Thus, the majority of CERV 2 elements were biologically active after the divergence of chimpanzees and humans from a common ancestor. Phylogenetic analysis of solo LTRs and LTRs from full-length elements revealed that CERV 2 elements group into at least four subfamilies (bootstrap values >95; Figure 4). We esti- mated the ages of two of the more abundant subfamilies by calculating the average of the pairwise distances between all sequences in each subfamily. The estimated ages of the two subfamilies were 21.9 MY and 14.1 MY, respectively. As was the case for the CERV 1/PTERV1 family, these age estimates are inconsistent with the fact that no CERV 2 orthologues were detected in the sequenced human genome. Again, we were able to detect pre-integration sites at those regions in the human genome orthologous to the CERV 2 insertion sites in chimpanzees, effectively eliminating the possibility that the elements were once present in humans but subsequently excised. We assessed the distribution of CERV 2 elements in primates by PCR using primers complementary to sequences in the conserved RT region. The results indicate that CERV 2 ele- ments are present in chimpanzee, bonobo and gorilla but absent in human, orangutan, old world monkeys, new world monkeys and prosimians (Figure 5a). Southern hybridization Phylogenetic tree of CERV 1/PTERV1 LTRsFigure 3 Phylogenetic tree of CERV 1/PTERV1 LTRs. Unrooted neighbor joining phylogenetic tree built from 5' and 3' LTRs from full-length CERV 1/ PTERV1 elements. The average pairwise distances (corrected 'p' using Jukes-Cantor model) for each subfamily and the estimated ages are shown. Bootstrap values are shown. 0.01 99 Subfamily 1 Average Pairwise distance : 2.5 % Estimated age: 7.8 MY Subfamily 2 Average Pairwise distance : 1.6% Estimated age: 5.0 MY R51.8 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, 7:R51 experiments were carried out on DNA from species that gave negative PCR results to eliminate the possibility that the PCR primer binding sites have diverged in distantly related species within the CERV 2 RT and gag regions complementary to the designed probes (Figure 5b). The combined PCR and South- ern analysis indicate that CERV 2 like sequences are present in chimpanzee, bonobo, gorilla and old world monkeys but absent in human, orangutan, new world monkeys and prosimians (Figure 5c). This distribution of CERV 2 elements among primates is identical to the above described distribution of CERV 1/PTERV1 elements [30]. It is worth noting that although the probes used in Southern hybridiza- tion were designed from chimpanzee element sequence, the strength of hybridization is higher in old world monkeys than in chimpanzees (Figure 5b), suggesting a higher copy number Phylogenetic tree of CERV 2 LTRsFigure 4 Phylogenetic tree of CERV 2 LTRs. Unrooted neighbor joining phylogenetic tree built from CERV 2 solo LTRs and 5' and 3' LTRs from full-length elements. The average pairwise distances (corrected 'p' using Jukes-Cantor model) for each subfamily and the estimated ages are shown. Bootstrap values are shown. 0.02 99 100 96 Subfamily 1 Average pairwise distance : 4.5% Estimated age : 14.1 MY Subfamily 2 Average pairwise distance : 7.2% Estimated age : 21.9 MY Subfamily 3 Subfamily 4 http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. R51.9 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2006, 7:R51 of CERV 2 elements in old world monkeys than in chimpanzees. Endogenous retroviral positional variation between chimpanzees and humans Comparative analyses of orthologous regions of the human and chimpanzee genomes has revealed a number of instances where relatively large spans of sequence present in one spe- cies are not present in the other [34,35]. It has been proposed that these gaps or INDELs may be of evolutionary signifi- cance (for example, [9]). To determine the proportion of these gaps (human gaps are sequences present in chimpan- zees but absent in humans; chimpanzee gaps are sequences present in humans but absent in chimpanzees) involving endogenous retroviruses, we utilized the human gap and chimpanzee gap datasets available at the UCSC Genome Bio- informatics web site [36] that were generated by aligning the chimpanzee genome with the human genome build HG16 [37,38]. These datasets include gaps of sizes ranging from 80 bp to 12.0 kb. Gap sequences from the datasets >5,000 bp (1,330 sequences), the typical length of full-length LTR retro- transposons/retroviruses, were blasted against the NCBI non-redundant protein database [39] using BlastX [40]. BLAST was used to identify species-specific full-length endogenous retroviral insertions in humans and chimpan- zees. A total of 41 chimpanzee gap sequences and 31 human gap sequences were found to have significant similarity (e < 0.01) with retroviral sequences. The presence of an endogenous retroviral sequence in chim- panzees that is missing at an orthologous genomic position in humans can be due to a novel insertion in chimpanzees or deletion of the element in humans. Similarly, the presence of an endogenous retroviral sequence in humans that is missing at an orthologous genomic position in chimpanzees can be due to novel insertion in humans or due to deletion of the ele- ment in chimpanzees. Because endogenous retroviruses do not precisely excise from insertion sites [4], it is possible to distinguish between these two possibilities. If a region in humans orthologous to the position of an endogenous retroviral insertion in chimpanzees contains a remnant of endogenous retroviral sequence (for example, fragmented element or solo LTR), we score the gap as a deletion in humans. If the orthologous region contains no remnant of the endogenous retrovirus but the pre-integration genomic sequence can be clearly identified, we score the gap as an insertion in chimpanzees. The same rules apply for the anal- ogous dataset of the endogenous retroviral sequences present in humans but absent in chimpanzees. Of the 41 instances where an endogenous retroviral sequence is present in chimpanzees but lacking in humans, 29 were due to novel insertions in chimpanzees while 12 were deletions in humans (Tables 3 and 4; Figure 6a). Of the 31 instances where an endogenous retrovirus is present in humans but absent in chimpanzees, we found that 8 were due to novel insertions in humans while 23 were deletions in chimpanzees (Table 4; Figure 6b). Of the 29 novel insertions in chimpanzees, 25 belong to the CERV 1/PTERV1 family, 2 to the CERV 2 family, 1 to the CERV 3 (HERVS7 1) family and 1 to the CERV 30 (HERVK10) family whereas all the 8 novel insertions in humans belong to the CERV 30 (HERVK10) family (Tables 3 and 4). Thus, four families of endogenous retroviruses have been transpositionally active in the chimpanzee lineage, resulting in full-length insertions, since chimpanzees and humans diverged from a common ancestor while only one of these families (CERV 30 (HERVK10)) has been active in humans (Tables 3 and 4). However, the family that is active in both humans and chimpanzees (CERV 30 (HERVK10)) gen- erated eight novel full-length insertions in humans as opposed to only one novel insertion in chimpanzees since they diverged from the common ancestor (Tables 3 and 4). Since solo LTRs and fragmented endogenous retroviral cop- ies are typically ten to a hundred times more abundant than full-length elements in humans [14,41], we extended our survey to determine the extent to which INDEL variation between humans and chimpanzees is associated with solo LTRs and/or fragmented endogenous retroviral sequences. We again utilized datasets (human gaps and chimp gaps) available at the UCSC Genome Bioinformatics web site [36]. We used 'Repeat Masker' (AF Smit and P Green, unpublished data) to identify all interspersed repeats, that is, all transpos- able elements present in the datasets, and to subsequently extract endogenous retroviral homologous sequences. Gap sequences were divided into two types: 'Mosaic type' gap sequences are defined as those composed of more than one category of interspersed repeats (for example, endogenous retrovirus inserted within a LINE element); and 'Single type' gap sequences are defined as those composed of only sequences homologous to endogenous retroviruses. Single type gap sequences were further divided into two categories: category 1 comprises those gap sequences composed entirely of an endogenous retroviral sequence; and category 2 com- prises those gap sequences composed of endogenous retrovirus and non-interspersed repeat sequences. The above categorizations are useful in distinguishing gaps due to dele- tions in one species from the gaps due to insertions in the other species. Instances of mosaic type and single type cate- gory 2 gaps are deletions in that species while the gaps that belong to single type category 1 are either deletions in that species or insertions in the other species. Because endog- enous retroviruses do not excise precisely [4] from the inser- tion sites, these later gaps can be further characterized as the result of insertions or deletions. We found a total of 18,395 human gap sequences of which 9,855 (53.57%) contained interspersed repeats. Chimpanzees had a total of 27,728 gap sequences of which 15,652 (56.44%) contained interspersed repeats. A total of 1,495 human gap sequences contained endogenous retroviral sequences (592 R51.10 Genome Biology 2006, Volume 7, Issue 6, Article R51 Polavarapu et al. http://genomebiology.com/2006/7/6/R51 Genome Biology 2006, 7:R51 Distribution of CERV 2 elements among primatesFigure 5 Distribution of CERV 2 elements among primates. Species surveyed include human (Homo sapiens), chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), crab eating monkey (Macaca fascicularis), rhesus monkey (Macaca mulatto), pig tailed monkey (Macaca nemestrina), black headed spider monkey (Ateles geoffroyi), wooly monkey (Lagothrix lagotricha), red-chested mustached tamari (Saguinus labiatus), and ring- tailed lemur (Lemur catta). (a) PCR was conducted using primers designed in the RT region of chimpanzee CERV 2 element. The PCR results indicate that the CERV 2 element is present in chimpanzee, bonobo, gorilla and absent in other primates. (b) Southern hybridization was carried out on the DNA of the primates with negative PCR results using a probe designed in the RT region. The results indicate that CERV 2 like elements are present in chimpanzee, crab eating macaque, rhesus monkey and pig tailed monkey. Though the same amount of DNA was loaded in all lanes, the strength of hybridization is higher in old world monkeys than in chimpanzees, suggesting a higher copy number of CERV 2 elements in old world monkeys than in chimpanzees. Below the figure, a restriction map (chimpanzee sequence from chromosome 5 position 53871447 53880194 (NCBI build 1 version 1)) is presented in relation to the hybridization probe, HindIII (triangles). (c) The results from the combined PCR and Southern analyses demonstrate a patchy distribution of CERV 2 elements among primates. Ladder Human Chimp Bonobo Gorilla Orangutan Macaque (Crab eating, Rhesus, Pig tailed macaque) - + + + - + ~ 7 Mya ~6 Mya ~ 12 Mya ~ 25 Mya Old World Monkeys Monkey (Black headed spider, wooly monkey) ~ 35 Mya New World Monkeys - Tamarin - Lemur - ~ 60 Mya Prosimians Human Chimp Bonobo Gorilla Oragutan Crab eating macaque Rhesus Monkey Pig tailed monkey Black headed spider monkey Wooly monkey Tamarin Lemur Negative Ladder Human Chimp Oragutan Crab eating macaque Rhesus Monkey Pig tailed monkey Black headed spider monkey Wooly monkey Tamarin Lemur Negative (a) (b) (c) 4920 bp RT probe 500 bp 600 bp 700 bp 800 bp 5.0 kb 6.0 kb 8.0 kb 4.0 kb 3.0 kb 2.0 kb 1.5 kb 1.0 kb [...]... mosaic type and 903 single type (640 category 1 + 263 category 2); Table 5) Five hundred and ninety two mosaic types and 263 single type category 2 were deletions in humans Of the 640 single type gaps belonging to category 1, 152 are new insertions in chimpanzees while the remaining 488 are deletions in humans Of the 152 chimpanzee insertions, 97 involved the two chimpanzee specific families while the remaining... mosaic types and 293 single type category 2 are deletions in chimpanzees Of the 557 that belonged to single type category 1, 79 are new insertions in humans while the remaining 478 are deletions in chimpanzees (Table 5) Consistent with the copy number of CERV 30 (HERVK10) species-specific full-length insertions (Table 4), the insertions of solo LTRs from this family were higher in humans (78) than in chimpanzees... sequences of little or no adaptive significance [42,43], transposable elements are today generally recognized as significant contributors to human regulatory (for example, [44]) and structural (for example, Genome Biology 2006, 7:R51 information A total of 1,608 chimpanzee gap sequences contained endogenous retroviral sequences (758 mosaic type and 850 single type (557 category 1+ 293 category 2) As... Figure 6 Structure of endogenous retroviral INDEL sequences (>5,000 bp) in humans and chimpanzees Structure of endogenous retroviral INDEL sequences (>5,000 bp) in humans and chimpanzees (a) Characteristics of the remnant endogenous retroviral sequences (solo LTRs and/ or fragmented elements) in humans The asterisk indicates 29 chimpanzee specific endogenous retroviral insertions of which: 25 belong... sequencing of the chimpanzee genome is providing a unique opportunity to conduct comparative genomic analyses of primate transposable elements Retrotransposons are the most abundant class of transposable elements For example, retrotransposons comprise at least 60% of the human genome [3] and results presented here and elsewhere [34] suggest that the number of endogenous retroviruses in chimpanzees may be... chimpanzee endogenous retroviruses range in age from about 0.8 to 145 MY Nine families of chimpanzee endogenous retroviruses have been transpositionally active in chimpanzees while two families of human endogenous retroviruses have been transpositionally active in humans since they diverged from a common ancestor (Table 5) Thus, while some families of endogenous retroviruses have not been transpositionally... attributable to the presence or absence of endogenous retroviral sequences The potential biological/evolutionary significance of this variation is currently under investigation Emerging evidence indicates that retrotransposons have played a significant role in gene and genome evolution (for example, [8-10]) The identification, characterization and comparative genomics of chimpanzee endogenous retroviruses presented... 1* - *Solo LTRs and/ or fragmented copies ln, length sequence over a stretch of 14 to 22 bp was assigned as a tRNA primer of the element (Table 2) 72°C and a final 1-cycle extension of 7 minutes at 72°C The PCR products were then visualized on 1% (w/v) agarose gel Evolutionary analysis of CERV 1/PTERV1 and CERV 2 LTR sequences Southern hybridization Molecular analysis: PCR and Southern hybridization Primate... restriction enzyme digested, transferred to a nylon membrane and hybridized as described previously [54] Nested PCR amplified products in RT and gag regions of CERV 2 elements were radioactively labeled and used as probes for hybridization The same amount of DNA was loaded in all the lanes, with DNA samples in the order: human (Homo sapiens), chimpanzee (Pan troglodytes), orangutan (Pongo pygmaeus), crab... 274:765-768 Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al.: Initial sequencing and analysis of the human genome Nature 2001, 409:860-921 Boeke JD, Stoye JP: Retrotransposons, endogenous retroviruses, and the evolution of retroelements In Retroviruses Edited by: Coffin JM, Hughes SH, Varmus H Plainview, NY: Cold Spring Harbor Laboratory Press; . characterization and comparative genomics of chimpanzee endogenous retroviruses Nalini Polavarapu, Nathan J Bowen and John F McDonald Address: School of Biology, Georgia Institute of Technology, Atlanta,. old world monkeys than in chimpanzees. Endogenous retroviral positional variation between chimpanzees and humans Comparative analyses of orthologous regions of the human and chimpanzee genomes. macaque) - + + + - + ~ 7 Mya ~6 Mya ~ 12 Mya ~ 25 Mya Old World Monkeys Monkey (Black headed spider, wooly monkey) ~ 35 Mya New World Monkeys - Tamarin - Lemur - ~ 60 Mya Prosimians Human Chimp Bonobo Gorilla Oragutan Crab